Modulation of the Pol II CTD Phosphorylation Code by Rac1 and Cdc42 Small GTPases in Cultured Human Cancer Cells and Its Implication for Developing a Synthetic-Lethal Cancer Therapy

Rho GTPases, including Rho, Cdc42, Rac and ROP subfamilies, are key signaling molecules in RNA polymerase II (Pol II) transcriptional control. Our prior work has shown that plant ROP and yeast Cdc42 GTPases similarly modulate Ser2 and Ser5 phosphorylation status of the C-terminal domain (CTD) of the Pol II largest subunit by regulating CTD phosphatase degradation. Here, we present genetic and pharmacological evidence showing that Cdc42 and Rac1 GTPase signaling modulates a similar CTD Ser2 and Ser5 phosphorylation code in cultured human cancer cells. While siRNA knockdown of Cdc42 and Rac1, respectively, in HeLa cells increased the level of CTD Ser phosphatases RPAP2 and FCP1, they both decreased the level of CTD kinases CDK7 and CDK13. In addition, the protein degradation inhibitor MG132 reversed the effect of THZ1, a CDK7 inhibitor which could decrease the cell number and amount of CDK7 and CDK13, accompanied by a reduction in the level of CTD Ser2 and Ser5 phosphorylation and DOCK4 and DOCK9 (the activators for Rac1 and Cdc42, respectively). Conversely, treatments of Torin1 or serum deprivation, both of which promote protein degradation, could enhance the effect of THZ1, indicating the involvement of protein degradation in controlling CDK7 and CDK13. Our results support an evolutionarily conserved signaling shortcut model linking Rho GTPases to Pol II transcription across three kingdoms, Fungi, Plantae and Animalia, and could lead to the development of a potential synthetic-lethal strategy in controlling cancer cell proliferation or death.

Fusion expression of recombinant human beta-defensin-3 and analysis of its biological activity

Human beta-defensins (hBDs) are small cationic antimicrobial peptides with multiple biologic activities. The aim of the study was cloning, expression in E.coli, purification and in vitro analysis of biological activity of recombinant human beta-defensin-3 (rec-hBD-3). hBD-3 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E.coli BL21(DE3) cells. Rec-hBD-3 was expressed in bacterial cells as GST-hBD-3 fusion protein, and purified by 3-step procedure via affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography on Sep-Pack C18. Analysis of biological activity of rec-hBD-3 has shown that the peptide is active against Pseudomonas aeruginosa in micromolar concentrations in radial diffusion test. Rec-hBD-3 did not affect proliferation and viability of cultured human cancer cells of A431, A549, and TPC-1 lines, but was capable to potentiate cytotoxic effects of rec-hBD-2 and docetaxel in vitro.